Method and apparatus for desorption and ionization of analytes

ABSTRACT

This invention relates generally to methods and apparatus for desorption and ionization of analytes for the purpose of subsequent scientific analysis by such methods, for example, as mass spectrometry or biosensors. More specifically, this invention relates to the field of mass spectrometry, especially to the type of matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry used to analyze macromolecules, such as proteins or biomolecules. Most specifically, this invention relates to the sample probe geometry, sample probe composition, and sample probe surface chemistries that enable the selective capture and desorption of analytes, including intact macromolecules, directly from the probe surface into the gas (vapor) phase without added chemical matrix.

[0001] The United States government has a paid-up license in thisinvention and the right in limited circumstances to require the patentowner to license others on reasonable terms as provided for by the termsof Grant No. 58-6250-1-003 awarded by the United States Department ofAgriculture.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention.

[0003] This invention relates generally to methods and apparatus fordesorption and ionization of analytes for the purpose of subsequentscientific analysis by such methods, for example, as mass spectrometryor biosensors. More specifically, this invention relates to the field ofmass spectrometry, especially to the type of matrix-assisted laserdesorption/ionization, time-of-flight mass spectrometry used to analyzemacromolecules, such as proteins or biomolecules.

[0004] 2. Description of the Prior Art.

[0005] Generally, analysis by mass spectrometry involves thevaporization and ionization of a small sample of material, using a highenergy source, such as a laser, including a laser beam. The material isvaporized from the surface of a probe tip by the laser beam, and in theprocess, some of the individual molecules are ionized by the gain of aproton. The positively charged ionized molecules are then acceleratedthrough a short high voltage field and let fly into a high vacuumchamber, at the far end of which they strike a sensitive detectorsurface. Since the time of flight is a function of the mass of theionized molecule, the elapsed time between ionization and impact can beused to determine the molecule's mass which, in turn, can be used toidentify the presence or absence of known molecules of specific mass.

[0006] All known prior art procedures which present proteins or otherlarge biomolecules on a probe tip for laser desorption/ionizationtime-of-flight mass spectrometry rely on a crystalline solid mixture ofthe protein or other analyte molecule in a large excess of acidic matrixmaterial deposited on the bare surface of a metallic probe tip. (Thesample probe tip typically is metallic, either stainless steel, nickelplated material or platinum). Immobilizing the analyte in such a matrixwas thought to be necessary in order to prevent the destruction ofanalyte molecules by the laser beam. The laser beam strikes the mixtureon the probe tip and its energy is used to vaporize a small portion ofthe matrix material along with some of the embedded analyte molecules.Without the matrix, the analyte molecules are easily fragmented by thelaser energy, so that the mass, and identity, of the originalmacromolecule is very difficult to determine.

[0007] This prior art procedure has several limitations which haveprevented its adaptation to automated protein or other macrobiologicalmolecules analysis. First, in a very crude sample it is necessary topartially fractionate (or otherwise purify the sample as much aspossible) to eliminate the-presence of excessive extraneous materials inthe matrix/analyte crystalline mixture. The presence of large quantitiesof components may depress the signal of the targeted analyte. Suchpurification is time-consuming and expensive and would be very difficultto do in an automated analyzer.

[0008] Second, while the amount of analyte material needed for analysisby the prior art method is not large (typically in a picomole range), insome circumstances, such as tests on pediatric patients, analyte fluidsare available only in extremely small volumes (microliters) and may beneeded for performing several different analyses. Therefore, even thesmall amount needed for preparation of the analyte/matrix crystallinemixture for a single analysis may be significant. Also, only a tinyfraction (a few thousandths or less) of analyte used in preparing theanalyte/matrix mixture for use on the probe tip is actually consumed inthe mass spectrometry analysis. Any improvement in the prior artprocedure which made it possible to use much less analyte to conduct thetest would be highly advantageous in many clinical areas.

[0009] Third, the analyte protein, or other macromolecule, used inpreparing the analyte matrix for use on the probe tip is not suitablefor any subsequent chemical tests or procedures because it is bound upin the matrix material. Also, all of the matrix material used to date isstrongly acidic, so that it would affect many chemical reactions whichmight be attempted on the mixture in order to modify the analytemolecules for subsequent examination. Any improvement in the procedurewhich made it possible to conduct subsequent chemical modifications orreactions on the analyte molecules, without removing them from thematrix or the probe tip, would be of enormous benefit to researchers andclinicians.

[0010] Additional limitations in the prior art included problems withmatrix use such as:

[0011] (1) formation of analyte-matrix complex (referred to as “matrixadduct” which interferes with the accuracy of analyte measurement;

[0012] (2) inability to wash away contaminants present in analyte ormatrix (e.g., other proteins or salts);

[0013] (3) formation of analyte-salt ion adducts;

[0014] (4) less than optimum solubility of analyte in matrix;

[0015] (5) signal (molecular ion) suppression “poisoning” due tosimultaneous presence of multiple components; and

[0016] (6) selective analyte desorption/ionization.

[0017] There are a number of problems and limitations with the prior artmethods. Prior investigators, including Karas and Hillenkamp havereported a variety of techniques for analyte detection using massspectroscopy, but these techniques suffered because of inherentlimitations in sensitivity and selectivity of the techniques,specifically including limitations in detection of analytes in lowvolume, undifferentiated samples. The “Hillenkamp-Karas” articles thatpertain to this field of invention are:

[0018] 1. Hillenkamp, “Laser Desorption Mass Spectrometry: Mechanisms,Techniques and Applications”; Bordeaux Mass Spectrometry ConferenceReport, 1988, pages 354-362.

[0019] 2. Karas and Hillenkamp, “Ultraviolet Laser Desorption ofProteins Up to 120,000 Daltons”, Bordeaux Mass Spectrometry ConferenceReport, 1988, pages 416, 417.

[0020] 3. Karas and Hillenkamp, “Laser Desorption Ionization of ProteinsWith Molecular Masses Exceeding 10,000 Daltons”, Analytical Chemistry,60. 2299, July 1988.

[0021] 4. Karas, Ingendoh, Bahr and Hillenkamp, “UV-LaserDesorption/Ionization Mass Spectrometry of Femtomol Amounts of LargeProteins”, Biomed. Environ. Mass Spectrum (in press).

[0022] The use of laser beams in time-of-flight mass spectrometers isshown, for example, in U.S. Pat. Nos. 4,694,167; 4,686,366, 4,295,046,and 5,045,694, incorporated by reference.

[0023] The first successful molecular mass measurements of intactpeptides and small proteins (only up to about 15 kDa) by any form ofmass spectrometry were made by bombarding surfaces with high energyparticles (plasma desorption and fast atom bombardment massspectrometry); this breakthrough came in 1981 and 1982. Improvementscame in 1985 and 1986, however, yield (signal intensities), sensitivity,precision, and mass accuracy remained relatively low. Higher molecularmass proteins (about 20 to 25 kDa) were not observed except on rareoccasions; proteins representing average molecular weights(approximately 70 kDa) were not ever observed with these methods. Thus,evaluation of most proteins by mass spectrometry remains unrealized.

[0024] In 1988, Hillenkamp and his coworkers used UV laser desorptiontime-of-flight mass spectrometry and discovered that when proteins ofrelatively high molecular mass were deposited on the probe tip in thepresence of a very large molar excess of an acidic, UV absorbingchemical matrix (nicotinic acid) they could be desorbed in the intactstate. This new technique is called matrix-assisted laserdesorption/ionization (MALDI) time-of-flight mass spectrometry. Notethat laser desorption time-of-flight mass spectrometry (without thechemical matrix) had been around for some time, however, there waslittle or no success determining the molecular weights of large intactbiopolymers such as proteins and nucleic acids because they werefragmented (destroyed) upon desorption. Thus, prior to the introductionof a chemical matrix, laser desorption mass spectrometry was essentiallyuseless for the detection of specific changes in the mass of intactmacromolecules (see below). Note that the random formation of matrixcrystals and the random inclusion of analyte molecules in the solidsolution is prior art.

SUMMARY OF THE INVENTION

[0025] The primary object of the invention is to provide improvedmethods, materials composition and apparatus for coupled adsorption,desorption and ionization of multiple or selected analytes into the gas(vapor) phase, preferably for use in conjunction with mass spectrometryof biomolecules and other macromolecules, as well as by means ofanalytic detection other than mass spectrometry. The invention incudes aflexible variety of options for presenting surfaces with attached energyabsorbing molecules, defined reaction sites, and affinity reagents forthe capture,e transfer, and/or the desorption of analytes before andafter a sseries of chemical, physical, and/or enzymatic modificationsperformed in situ.

[0026] Another object is to provide such a method and apparatus foraffinity-directed detection of analytes, including desorption andionization of analytes in which the analyte is not dispersed in a matrixsolution or crystalline structure but is presented within, on or abovean attached surface of energy absorbing “matrix” material throughmolecular recognition events, in a position where it is accessible andamenable to a wide variety of chemical, physical and biologicalmodification or recognition reactions.

[0027] The probe surface with and without bonded energy absorbingmolecules, referred to as the sample presenting surface can be composedof a variety of materials, including porous or nonporous materials, withthe porous materials providing sponge-like, polymeric, high surfaceareas for optimized adsorption and presentation of analyte.

[0028] These surface materials can be substituted (at varying densities)with chemically bonded (covalent or noncovalent) affinity adsorptionreagents and/or chemically bonded (i.e., immobilized) energy absorbingmolecules (bound “matrix” molecules). The geometry of the samplepresenting surface can be varied (i.e., size, texture, flexibility,thickness, etc.) to suit the need (e.g., insertion into a livingorganism through spaces of predetermined sizes) of the experiment(assay).

[0029] Another object is to provide such a method and apparatus in whichthe analyte material is chemically bound or physically adhered to asubstrate forming a probe tip or other sample presenting surface.

[0030] A further object is to provide means for the modification ofprobe or sample presenting surfaces with energy-absorbing molecules toenable the successful desorption of analyte molecules without theaddition of exogenous matrix molecules as in prior art.

[0031] A further object is to provide the appropriate density ofenergy-absorbing molecules bonded (covalently or noncovalently) in avariety of geometries such that mono layers and multiple layers ofattached energy-absorbing molecules can be used to facilitate thedesorption of analyte molecules of varying masses. The optimum ratio ofadsorbed or bonded energy-absorbing molecules to analyte varies with themass of the analyte to be detected. A further object is to modify thesample presenting surface with such energy-absorbing molecules where thecomposition of the probe or sample presenting surface is other than themetal or metallic surfaces as described in prior art. Separate from thechemical and/or physical modification of the probe surface with energyabsorbing molecules is the modification-of-these surfaces with affinityreagents, both chemical and/or biological, for the specific purpose ofcapturing (adsorbing) specific analyte molecules or classes of analytemolecules for the subsequent preparation, modification, and successfuldesorption of said analyte molecules.

[0032] A further object is to provide all combinations of surfacesmodified with energy-absorbing molecules and/or affinity-directedanalyte capture devices to enable the selective and/or nonselectiveadsorption of analytes and the subsequent desorption either with orwithout requiring the subsequent addition of additional matrixmolecules. It is important to note that the surfaces modified withaffinity reagents for the capture of analytes are-more useful than theunderivitized sample surfaces described in prior art even when thedeposition of energy-absorbing molecules (that is matrix) is asdescribed in prior art. Because of the advantages in the ability toremove contaminating substances from the adsorbed analyte molecules andbecause of the ability to modify adsorbed analyte molecules without (orbefore) added matrix.

[0033] A further object is to provide such a method and apparatus inwhich the substrate forming the probe tip or other sample presentingsurface is derivatized with one or more affinity reagents (a variety ofdensities and degrees of amplification) for selective bonding withpredetermined analytes or classes of analytes.

[0034] A further object is to provide methods and apparatus for usingprobe tips having surfaces derivatized with affinity reagents andcontaining laser desorption matrix material (chemically bonded tosurface or not) which may be used to isolate target analyte materialsfrom undifferentiated biological samples such as blood, tears, urine,saliva, gastrointestinal fluids, spinal fluid, amniotic fluid, bonemarrow, bacteria, viruses, cells in culture, biopsy tissue, plant tissueor fluids, insect tissue or fluids, etc.

[0035] Because of the new and preferred method for presentation anddesorption of selected analytes, a further object is to use analytedetection methods other than the generic electron multipliers typicallyused in mass spectrometric devices. This would include but would not belimited to detection films/plates for the qualitative or quantitativeevaluation of fluorescent or radio-labeled anialytes or analyte moleculecomplexes.

[0036] A further object is to provide such a system in which theaffinity reagent chemically bonds or biologically adheres to the targetanalyte or class of analytes.

[0037] A further object is to use existing and new solid phase affinityreagents (e.g., small diameter porous or nonporous beads ofcrosslineated polymer with attached molecular capture devices) designedfor the (1) capture (adsorption of one or more analytes, (2) thepreparation of these captured analytes (e.g., washing with H₂O or otherbuffered or nonbuffered solutions to remove contaminants such as salts,multiple cycles of washing, such as with polar organic solvent,detergent-dissolving solvent, dilute acid, dilute base or urea), and (3)most importantly, the direct transfer of these captured and preparedanalytes to the probe surface for subsequent analyte desorption (fordetection, quantification and/or mass analysis).

[0038] A further object is to provide such a system in which thepredetermined analytes are individual biomolecules or othermacromolecules or combinations of adjoined molecules (i.e., complexes).

[0039] A still further object is to provide such a method and apparatusin which the matrix materials used are not strongly acidic, as in priorart matrices, but are chemically modified into the slightly acidic,neutral pH or strongly basic range of pH.

[0040] A further object is to provide such a system in which the matrixmaterial has a pH above 6.0.

[0041] A still further object is to provide a method and apparatus fordesorption and ionization of analytes in which unused portion of theanalytes contained on the presenting surface remain chemicallyaccessible, so that a series of chemical and/or enzymatic or othertreatments (e.g., discovery of analyte-associated molecules by molecularrecognition) of the analyte may be conducted on the probe tip or otherpresenting surface, in situ, followed by sequential analyses of themodified analyte by mass spectrometry. In one case (i.e., repetitivesequential analyses) the analyte is adsorbed to the sample presentingsurface and can be treated (modified in situ after the excess freematrix is removed (i.e., washed away). Matrix can be added back beforeanalysis by mass spectrometry. Using this procedure, an analyte can berepeatedly tested for a variety of components by removing one matrix,modifying the analyte sample, re-applying the same or different matrix,analyzing the sample, etc.

[0042] A further object is to provide a method and apparatus for thecombined chemical and/or enzymatic modifications of target analytes forthe purpose of elucidating primary, secondary, tertiary, or quaternarystructure of the analyte and its components.

[0043] A still further object is to provide such a method and apparatusin which the probe tips or other sample presenting surfaces are formedof a variety of materials, including electrically insulating materials(porous and nonporous), flexible or nonrigid materials, opticallytransparent materials (e.g., glass, including glass of varyingdensities, thicknesses, colors and with varying refractive indices), aswell as less reactive, more biocompatible materials (e.g., biopolymerssuch as agarose, dextran, cellulose, starches, peptides, and fragmentsof proteins and of nucleic acids such as DNA (deoxyribonucleic acid) andRNA (ribonucleic acid). These surfaces can be chemically modified by theattachment of energy-absorbing molecules and/or affinity directedanalyte capture molecules.

[0044] Another object is to provide a method and apparatus fordesorption and ionization of analyte materials in which cations otherthan protons (H⁺) are utilized for ionization of analyte macromolecules.

[0045] Note that the laser or light source used to convey energy to theprobe surface can employ a wavelength(s) that is(are) not fixed but canbe varied according to the wavelength absorbed by the matrix (whetherthe matrix is added in the free form or is chemically bonded to theprobe (sample presenting) surface). For this procedure, a variety ofwavelengths (10 or more) defined by absorbance of matrix or energyabsorbing surface can be utilized.

[0046] Another object is to provide such a method and apparatus in whichthe probe tips or other sample presenting surfaces used for laserdesorption/ionization time-of-flight mass spectrometry are magnetizedand in which the matrix, affinity directed absorption molecules and/oranalyte materials are magnetically adhered to such magnetized surface.

[0047] A further object is a method and apparatus in which the matrixand/or analyte materials are adhered by any variety of chemicalmechanisms to the sample presenting surface.

[0048] A further objective is to provide energy-absorbing moleculeswhich have been incorporated into other chemical structures (e.g.,chemical or biological polymers) for the deposition (covalent ornoncovalent) onto the sample presenting surface in a way that enablesrepetitious analyte desorption events without interference with chemicaland/or enzymatic modifications of the analyte molecule(s) performed insitu.

[0049] Another object is to provide sample presenting surfaces in avariety of sizes and configurations (up to 4“×4”) with multiple (up to10,000 or more) spots (including spots down to <0.001 inch diameter) ofaffinity reagents arranged in predetermined arrays for the selectiveadsorption of numerous different analytes (e.g., clinical chemicalmarker proteins) to enable a wide spectrum sampling of themacromolecular composition of biological samples/fluids.

[0050] As shown more fully below, the present invention overcomeslimitations and disabilities of the prior art by providing probe tips orsample plates whose surfaces have been derivatized with biospecificaffinity reagents which will selectively bind specific groups or typesof biomolecules or other analytes out of an undifferentiated sample(such as blood or urine). Appropriate selection of the affinity reagentsused to derivatize the probe tip surface therefore makes possible theselection from the undifferentiated sample and binding to the probe tipof the specific types or groups of biological or other macromoleculesunder investigation, or subsequent examination (e.g., quantificationand/or structure elucidation) by mass spectrometry. This has theadvantage of achieving both the purification of the analyte samplepreviously required and the effect of concentrating the analyte. Itreduces by a factor of 1,000 to 100,000 the amount of analyte needed forthe mass spectrometry examination, since only the macromolecules whichattach to the biospecific affinity reagents are removed from the analytesample, and these can be sequestered on predetermined areas of the probetips or sample plates that are even less than the laser spot size.

[0051] It also has been found that the probe tips used in the process ofthe invention need not be metal or metal-coated, as with prior artprocedures. Research involved in the invention has involved glass andsynthetic polymer surfaces such as polystyrene, polypropylene,polyethylene, polycarbonate and other polymers including biopolymers,for the probe tips which have been covalently or noncovalentlyderivatized for immobilization of specific reagents that will direct theselective adsorption of specific analytes. These surfaces will includeimmobilized metal ions, immobilized proteins, peptides, enzymes, andinhibitor molecules, immobilized DNA and RNA, immobilized antibodies,immobilized reducing agents, immobilized carbohydrates and lectins,immobilized dyes and immobilized protein surface domains involved inmolecular recognition (e.g., dimerization domains and subunits). Some ofthe chemical and surface structures are as yet unknowvn.

[0052] The preferred probe tip, or sample plate, for selectiveadsorption/presentation of sample for mass analysis are (1) stainlesssteel (or other metal) with a synthetic polymer coating (e.g.,cross-linked dextran or agarose, nylon, polyethylene, polystyrene)suitable for covalent attachment of specific biomolecules or othernonbiological affinity reagents, (2) glass or ceramic, and/or (3)plastic (synthetic polymer). The chemical structures involved in theselective immobilization of affinity reagents to these probe surfaceswill encompass the known variety of oxygen-dependent, carbon-dependent,sulfur-dependent, and/or nitrogen-dependent means of covalent ornoncovalent immobilization. The methods and chemical reactions used inproducing such surfaces derivatized with biospecific affinity reagentsalready are known by those skilled in the art. Two features of theinvention, however, are (1) the specific size and localization of thederivatized surface with respect to the laser beam and (2) the affinitydirected presentation of specific analyte molecules (e.g., macromoleculeor biopolymer) at a defined surface density or local concentrationrequired for the efficient detection by laser desorption/ionizationtime-of-flight mass spectrometry. This can be accomplished by arrangingthe affinity adsorption “spots” (0.005 to 0.080 inch diameter) on theprobe surface in a defined manner (400 to 1,000 spots could be placed ona surface about the size of a glass slide).

[0053] An additional discovery involves the fact that pH modifiedchemical matrices can be used on these surfaces to facilitatedesorption/ionization without disruption of conditions necessary forsubsequent sample modification. As discussed above, prior art matrixmaterials used for biomolecular mass spectrometry are acidic. The exactchemical structure of the pH-modified matrices still are unknown.However, by suitable neutralization of the matrix material, it can bemade largely passive to subsequent chemical or enzymatic reactionscarried out on the analyte molecules presented on the derivatized probetip surface by the biospecific affinity reagents. This makes possiblethe carrying out of chemical reactions on the analyte moleculespresented on the probe tips. Since only a small fraction of the analytemolecules are used in each desorption/mass spectrometer measurement, anumber of sequential chemical and/or enzymatic modifications of thesamples, in situ, on the probe tips, and subsequent analysis of themodified samples by mass spectrometry, can be carried out on the sameprobe tips in order to more accurately determine exactly what moleculeis present, or other characteristics or information about the molecule,including its structure.

[0054] Finally, even when these matrix molecules are immobilized on aprobe tip surface, the analyte deposited on such a surface can bedesorbed with a laser beam. This circumvents the contamination of theanalyte by the matrix molecules. As a particular feature of theinvention, we have shown that some energy absorbing molecules that donot work as “matrix” molecules when added to analytes as a solution offree molecules (as in prior art) do indeed work well to facilitate thedesorption of intact analyte molecules after being immobilized.

[0055] It seems likely that these improvements in the procedure willenable bioanalytical and medical instrument manufacturers to develop amachine for the automated evaluation of a single protein sampledeposited on a surface and modified with numerous chemical and/orenzymatic reactions performed in situ.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The foregoing and other objects and advantages of the inventionwill be apparent from the following specification and from theaccompanying drawings.

[0057]FIG. 1A is a mass spectrum of peptide mixtures using sinapinicacid pH 6.5 as the matrix. FIG. 1B is a mass spectrum of the samepeptide mixtures after in situ addition of CuSO₄.

[0058]FIG. 2A is a mass spectrum of human casein phosphopeptide(R1-K18+5P) using sinapinic acid pH 6.5 as the matrix. FIG. 2B shows themass spectrum of the same peptide after in situ alkaline phosphatasedigestion for 5 and 10 min respectively. FIG. 2D shows the mass spectrumof the same peptide using dihydroxybenzoic acid pH 2 as the matrix,after in situ alkaline phosphatase digestion for 10 min.

[0059]FIG. 3 is a mass spectrum histidine-rich glycoprotein (GHHPH)₅Gpeptide using sinapinic acid pH 6.2 as the matrix before and after insitu digestion with carboxypeptidase P.

[0060]FIG. 4 is a composite mass spectra of peptide mixtures usingsinapinic acid as the matrix on glass, polypropylene-coated steel,polystyrene-coated steel and nylon probe tips.

[0061]FIG. 5A is a mass spectrum of peptides unadsorbed by TSKSW-IDA-Cu(II). FIG. 5B is a mass spectrum of peptide adsorbed by TSKSW-IDA-Cu(II). FIG. 5C is a mass spectrum of the same peptide adsorbedon TSK SW-IDA-Cu(II) after water wash.

[0062]FIG. 6 is a mass spectrum of myoglobin (<8 fmole)affinity-adsorbed on TSK SW-IDA-Cu(II).

[0063]FIG. 7A is a mass spectrum of proteins/peptides in infant formula.FIG. 7B is a mass spectrum of phosphopeptides in the same sampleaffinity-adsorbed on Sepharose-TED-Fe(III). FIG. 7C is a mass spectrumof proteins/peptides in gastric aspirate of preterm infant. FIG. 7D is amass spectrum of the phosphopeptides in the same sample adsorbed onSepharose-TED-Fe(III).

[0064]FIG. 8 (bottom) is a mass spectrum of rabbit anti-humanlactoferrin immunoglobin affinity adsorbed on paramagneticDynabead-sheep anti-rabbit IgG. FIG. 8 (top) is a mass spectrum of humanlactoferrin and rabbit anti-human lactoferrin IgG complex affinityadsorbed on paramagmetic Dynabead-sheep anti-rabbit IgG.

[0065]FIG. 9 is a mass spectrum of human lactoferrin affinity adsorbedon a single bead of agarose-single-stranded DNA deposited on a 0.5 mmdiameter steel probe tip.

[0066]FIG. 10 is the mass spectrum of human lactoferrin affinityadsorbed from urine on agarose-single-stranded DNA.

[0067]FIG. 11A is a mass spectrum of human gastrointestinal fluid. FIG.11B is a mass spectrum of trypsin in the same sample affinity adsorbedon AffiGel 10-soybean trypsin inhibitor.

[0068]FIG. 12A is a mass spectrum of human serum proteins. FIG. 12B is amass spectrum of human serum albumin in the same sample affinityadsorbed on agarose-Cibacron blue.

[0069]FIG. 13 is a drawing of the surface bound cinnamamide; Rrepresents the surface plus cross-linear.

[0070]FIG. 14A is a mass spectrum of peptide mixtures on surface boundcinnamamide. FIG. 14B shows the mass spectrum of the same peptidemixtures on free cinnamamide.

[0071]FIG. 15 is a drawing of the surface bound cinnamyl bromide; tvostructural forms are possible; R represents the surface pluscross-linear.

[0072]FIG. 16A is a mass spectrum of peptide mixtures on surface boundcinnamyl bromide. FIG. 16B is a mass spectrum of the same peptidemixtures on free cinnamyl bromide.

[0073]FIG. 17 is a drawing of the surface bound MAP-dihydroxybenzoicacid; R represents the surface plus cross-linear.

[0074]FIG. 18 is a mass spectrum of peptide mixtures on surface boundMAP alone (control surface). FIG. 18B is a mass spectrum of the samepeptide mixtures on surface bound MAP-dihydroxybenzoic acid.

[0075]FIG. 19A is a mass spectrum of myoglobin on surface boundcyanohydroxycinnamic acid. FIG. 19B is the same mass spectrum in the lowmass region.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Detailed Description

[0076] Usage of Conventional Matrix in Aqueous, pH-neutralized Form

EXAMPLES

[0077] Sinapinic acid (dimethoxy hydroxycinnamic acid) (Aldrich ChemicalCo., Inc., Milwaukee, Wis.) 20 mg/ml water suspension (intrinsic pH3.88) Dihydoxybenzoic acid (Aldrich) 20 mg/ml water (intrinsic pH 2.07)Cyanohydroxycinnamic acid (Aldrich) 20 mg/ml water suspension (intrinsicpH 3.3) each titrated with triethylamine (Pierce, Rockford, Ill.) to pH6.5, 7.2 and 6.5 respectively

[0078] 2 ul of the matrix solution was mixed with 1 ul of sample andallowed to air dry

[0079] 1. A mixture of synthetic peptides-human histidine-richglycoprotein peptide (GHHPH)₂G, (GHHPH)₅G, human estrogen receptordimerization domain (D473-L525) with neutralized sinapinic acid as thematrix, in the absence and presence of Cu(II). FIG. 1 showed the in situmetal-binding properties of the peptides under neutralized condition.

[0080] 2. Casein phosphopeptide (R1-K18+5P) with sinapinic acid pH 6.5.Followed by in situ alkaline phosphatase (0.5 ul, Sigma) digestion for10 min at room temperature. Similar in situ digestion on the samepeptide with dihydoxybenzoic acid (prepared in 30% methanol/0.1%trifluoroacetic acid) was used as control. FIG. 2 showed the moreefficient enzymatic dephosphorylation under neutralized condition.

[0081] 3. Mixture of synthetic peptides as in 1 with sinapinic acid pH6.2, followed by in situ carboxypeptidase P (1 ul, Boehringer MannheimCorp, Indianapolis, Id., 20 ug/50 ul) digestion for 30 min at roomtemperature. FIG. 3 showed preferential removal of C-terminal amino acidfrom histidine-rich glycoprotein peptide. Also showed unambiguousC-terminal determination even in peptide mixtures.

[0082] Usage of Probe Tip (Surface) Materials (Composition) other thanStainless Steel or Platinum for Sample Deposition

EXAMPLES

[0083] Molten polypropylene or polystyrene was deposited on stainlesssteel probe tip so as to cover it completely

[0084] Solid glass rod (1.5 mm dia) was cut into 1 cm segments andinserted into stainless steel probe tip support

[0085] Solid nylon (Trimmer line, 1.5 mm dia, Arnold, Shelby, Ohio) wascut into 1 cm segments and inserted into stainless steel probe tipsupport

[0086] Magnetic stir. bars (1.5×8 mm, teflon coated, Curtin MathesonScientific, Inc., Houston Tex.) inserted into stainless steel probe tipsupport

[0087] Peptide mixtures (as in FIG. 1 with dihydroxybenzoic acid in 30%methanol/0.1% TFA) on all four surfaces. FIG. 4

[0088] Affinity-directed Laser Desorption (with Matrix Added asDescribed in Prior Art)

EXAMPLES

[0089] Group 1. Immobilized Metal Ion as the Affinity Ligand

[0090] Cu(II) was chelated by iminodiacetate group covalently attachedto either porous agarose beads (Chelating Sepharose Fast Flow, PharmaciaBiotech Inc., Piscataway, N.J., ligand density 22-30 umole/ml gel) orsolid TSK-SW beads (ToyoSoda, Japan, ligand density 20 umole/ml gel)

[0091] Fe(III) was chelated bytris(carboxymethyl)ethylenediamine-Sepharose 6B (synthesized asdescribed by Yip and Hutchens, Protein Expression and Purification2(1991)355-362, ligand density 65 umole/ml)

[0092] 1. A mixture of synthetic peptides, neurotensin (30 nmole), spermactivating peptide (50 nmole) and angiotensin I (150 nmole), were mixedwith 50 ul packed volume of TSK SW-IDA-Cu(II) at pH 7.0 (20 mM sodiumphosphate, 0.5 M NaCI) at room temperature for 10 min. The gel was thenwashed with 3×200 ul sodium phosphate buffer, containing 0.5 M NaCl, pH7.0 and suspended in equal volume of water. 2 ul of the gel suspensionwas mixed with 1 ul sinapinic acid (methanol). FIG. 5A showed themolecular ions (and multiple Na-adducts) of neurotensin and spermactivating factor which were not adsorbed by the IDA-Cu(II). The massspectrum in FIG. 5B showed mainly the angiotensin I plus Na-adducts.When the IDA-Cu(II) gel was further washed with 500 ul of water 2×, theresulting mass spectrum showed only the parent angiotensin I specieswith no other adduct peaks. When the IDA-Cu(II) gel beads with adsorbedangiotensin was incubated with cyanohydroxycinnamic acid (20 mg/mlwater) pH 7.0 for 10 min at room temperature and then analyzedseparately, the angiotensin I was found to be still associated with thegel beads and not with the matrix solution.

[0093] 2. Horse heart myoglobin (325 pmole) was mixed with 50 ul of TSKSW-IDA-Cu(II) gel beads in 20 mM sodium phosphate, 0.5 M NaCl, pH 7.0 atroom temperature for 10 min. The gel beads were then washed with 2×500ul of buffer and 2×500 ul of water. The beads were suspended in equalvolume of water and then serial diluted into water. 0.5 ul of thediluted gel suspension was mixed with 1 ul of sinapinic acid (30%methanol/0.1% TFA). A detectable signal (after averaging 50 laser shots)of myoglobin was still obtained when the calculated quantity equivalentto or less than 8 fmole was deposited on the probe tip. FIG. 6

[0094] 3. 100 ul of infant formula and gastric content of preterm infantaspirated 90 min after feeding of infant formula was mixed with 50 ul ofTED-Fe(III) in 0.1 M MES, 0.15 M NaCl, pH 6.5 at room temperature for 15min. The gel beads were then washed with 3×500 ul of MES buffer and thenwith 1×500 ul of water. 1 ul of the gel suspension was mixed with 2 ulof sinapinic acid (50% acetonitrile/0.1% TFA). The result showed thatgastric aspirate had much more low molecular weight phosphopeptides(i.e., bound by TED-Fe(III)) than the formula due to proteolyticdigestion. In situ alkaline phosphatase digestion of peptides adsorbedon the TED-Fe(III) gel beads showed shifts to lower molecular weightindicating that they are indeed phosphopeptides. FIG. 7

[0095] Group 2. Immobilized Antibody as the Affinity Ligand

[0096] Polyclonal rabbit anti-human lactoferrin antibody was customgenerated for this lab by Bethyl Laboratories (Montgomery, Tex.). It waspurified by thiophilic adsorption and then by immobilized lactoferrincolumn. Sheep anti-rabbit IgG covalently attached to magnetic beads wereobtained from Dynal AS (Oslo, Norway) (uniform 2.8 um superparamagneticpolystyrene beads, ligand density 10 ug sheep IgG per mg bead).

[0097] 1. Human lactoferrin (1 nmole) was incubated with rabbitantihuman lactoferrin at 37° for 30 min. Subsequently, 40 ul of sheepanti-rabbit IgG on Dynabeads (6-7×10⁸ beads/ml) was added and incubatedat 37° for 30 min. The beads were then washed with 3×500 ul of sodiumphosphate buffer, and 2×500 ul of water. The final amount of humanlactoferrin bound to the complex was estimated to be 4 pmole.Approximately {fraction (1/10 )} of the beads was transferred to amagnetic probe tip and mixed with 2 ul of sinapinic acid (30% MeOH/0.1%TFA). Result showed the lactoferrin ion signal in addition to the rabbitIgG signal. FIG. 8

[0098] Group 3. Immobilized Nucleic Acid as the Affinity Ligand

[0099] Single-strand DNA immobilized on 4% agarose beads was obtainedfrom GIBCO BRL, Gaithersburg, Md. The ligand density was 0.5-1.0 mg/ml.

[0100] 1. 200 ul of ¹²⁵I human lactoferrin (equivalent to 49 nmole) wasmixed with 100 ul of immobilized single-strand DNA in 20 mM HEPES, pH7.0 at room temperature for 10 min. The beads were then washed with5×500 ul of HEPES buffer and then suspended in equal volume of water.The amount of lactoferrin bound per bead was found to be 62 fmole bydetermining the radioactivity and counting the number of beads per unitvolume. Various numbers of beads (from 1 to 12) were deposited on 0.5 mmdiameter probe tips and mixed with 0.2 ul of sinapinic acid (30%methanol/0.1% TFA). Lactoferrin ion signals were obtained with multiple100 laser shots on a single bead with adsorbed lactoferrin. FIG. 9

[0101] 2. 30 pmole of ⁵⁹Fe-human lactoferrin was added to 1 ml ofpreterm infant urine and mixed with 20 ul of immobilized single-strandDNA on agarose in 0.1 M HEPES pH 7.4 at room temperature for 15 min. Thebeads were washed with 2×500 ul HEPES buffer, and 2×500 ul of water. Thebeads were then suspended in equal volume of water and 1 ul (equivalentto not more than 350 fmole as determined by radioactivity) was mixedwith 1 ul sinapinic acid (30% methanol/0.1% TFA) on a probe tip.Positive lactoferrin signals were obtained for multiple 50 laser shots.FIG. 10

[0102] Group 4. Immobilized Biomolecule as the Affinity Ligand Soybeantrypsin inhibitor (Sigma, St Louis, Mo.) was immobilized on AffiGel 10(BioRad Laboratories, Hercules, Calif., ligand density 15 umole/ml)according to manufacturer's instructions.

[0103] 1. 100 ul of human gastrointestinal aspirate was mixed with 50 ulof immobilized soybean trypsin inhibitor in 20 mM sodium phosphate, 0.5M sodium chloride, pH 7, at room temperature for 15 min. The gel beadswere then washed with 3×500 ul of phosphate buffer, and 2×500 ul ofwater. 1 ul of gel bead suspension was mixed with 2 ul of sinapinic acid(50% acetonitrile/0.1% TFA). Result showed the presence of trypsin andtrypsinogen in the aspirate. FIG. 11

[0104] Group 5. Immobilized Dye as the Affinity Ligand

[0105] Cibacron Blue 3GA-agarose (Type 3000, 4% beaded agarose, liganddensity 2-5 umoles/ml, Sigma).

[0106] Other immobilized dyes include Reactive Red 120-agarose, ReactiveBlue-agarose, Reactive Green-agarose, Reactive Yellow-agarose (Sigma)

[0107] 1. 200 ul of human plasma was mixed with 50 ul of immobilized dyein 20 mM sodium phosphate, 0.5 M NaCl, pH 7.0 at room temperature for 10min. The gel beads were then washed with 3×500 ul of phosphate bufferand 2×500 ul of water. 1 ul of gel bead suspension was mixed with 2 ulof sinapinic acid (50% acetonitrile/0.1% TFA). Result showed theselective adsorption of human serum albumin from the serum sample byCibacron Blue. FIG. 12

[0108] Surface-enhanced Laser Desorption

EXAMPLES

[0109] Group 1. Energy-absorbing Molecule Covalently Bonded to Surfacevia the N-group

[0110] Cinnamamide (Aldrich, not a matrix by prior art) was dissolved inisopropanol/0.5 M sodium carbonate (3:1) and mixed with divinyl sulfone(Fluka, Ronkonkoma, N.Y.) activated Sepharose (Pharmacia) at roomtemperature for 2 hr. The excess molecules were washed away withisopropanol. The proposed structure was presented in FIG. 13. 2 ul ofbound or free molecule was deposited on the probe tips, 1 ul of peptidemixtures in 0.1% TFA was added on top and the result showed the peptideion signals detected only for the bound form. FIG. 14.

[0111] Group 2. Energy Absorbing Molecule Covalently Bonded to Surfacevia the C-group

[0112] Cynnamyl bromide (Aldrich, not a matrix by prior art) wasdissolved in isopropanol/0.5 M sodium carbonate and mixed with divinylsulfone-activated Sepharose at room temperature overnight. The excessmolecules were washed away with isopropanol. The proposed structures arepresented in FIG. 15. 2 ul of the bound or free molecule was depositedon the probe tip, 1 ul of peptide mixtures in 0.1% TFA was added on topand the result showed the detection of peptide ion signal only for thebound form. FIG. 16.

[0113] Group 3. Energy Absorbing Molecule Covalently Bonded to Surfacevia the C-group

[0114] Dihydroxybenzoic acid was activated by carbodiimide and mixedwith Fmoc-MAP 8 branch resin (Applied Biosystems, Forster City, Calif.)overnight. The proposed structure was presented in FIG. 17. Afterwashing, 1 ul of the bonded molecule on MAP or the MAP alone in 50%acetonitrile/0.1% TFA were deposited on the probe tip, 1 ul of peptidemixture was added on top, the resulting mass spectrum was presented inFIG. 21.

[0115] Group 4. Energy Absorbing Molecule Covalently Bonded to Surfacevia Undetermined Group.

[0116] Cyanohydroxycinnamic acid was dissolved in methanol and mixedwith AffiGel 10 (BioRad) at room temperature for two hours. The unboundmolecules were washed away with methanol. Protein samples that are foundto desorb successfully from this modified surface include myoglobin(FIG. 19), trypsin and carbonic anhydrase.

[0117] These examples (Groups 1-4) are also demonstrations of combinedsurface-enhanced and affinity-directed desorption where the adsorbed(bonded) energy absorbing molecular also act as affinity adsorptionreagents to enhance the capture of analyte molecules.

[0118] Definitions

[0119] (1) “Presenting surface”—the probe tip, sample plate or othersurface on which the analyte and matrix are presented fordesorption/ionization and analysis for example by mass spectrometry.

[0120] (2) “Matrix”—as described in prior art as the substance mixedwith the analyte (typically prior to deposition) and deposited on thepresenting surface in association with the analyte to absorb at leastpart of the energy from the energy source (e.g., laser) to facilitatedesorption of intact molecules of the analyte.

[0121] (3) “Analyte”—the material which is the subject of desorption andinvestigation by mass spectrometry or other means for detection.

[0122] (4) “Affinity reagent” (analyte capture device)—the class ofmolecules (both man made, unnatural, natural and biological) and/orcompounds which have the ability of being retained on the presentingsurface (by covalent bonding, chemical absorption, etc.) while retainingthe ability of recognition and bonding to an analyte.

[0123] (5) “Desorption”—the departure of analyte from the surface and/orthe entry of the analyte into a gaseous phase.

[0124] (6) “Ionization”—the process of creating or retaining on ananalyte an electrical charge equal to plus or minus one or more electronunits.

[0125] (7) “Adduct”—the appearance of an additional mass associated withthe analyte and usually caused by the reaction of excess matrix (ormatrix break-down products) directly with the analyte.

[0126] (8) “Adsorption”—the chemical bonding (covalent and/ornoncovalent) of the energy-absorbing molecules, the affinity reagent(i.e., analyte capture device), and/or the analyte to the probe(presenting surface).

What is claimed:
 1. An apparatus for measuring the mass of analytemolecules by means of mass spectrometry, said apparatus comprising: aspectrometer tube; vacuum means for applying a vacuum to the interior ofsaid tube; electrical potential means within the tube for applying anaccelerating electrical potential to desorbed analyte molecules; samplepresenting means removably insertable into said spectrometer means, forpresenting said analyte molecules in association with a matrix materialfor promoting desorption and ionization of said analyte molecules, saidsample presenting means being adapted to present said analyte moleculeson or above the surface of said matrix material, whereby at least aportion of said analyte molecules not consumed in said mass spectrometryanalysis will remain accessible for subsequent chemical analyticalprocedures; laser beam means for producing a laser beam directed to saidanalyte molecules and matrix material on said sample presenting meansinserted into said spectrometer means, for imparting sufficient energyto desorb and ionize a portion of said analyte molecules on said samplepresenting means; and detector means associated with said spectrometertube for detecting the impact of accelerated ionized analyte moleculesthereon.
 2. A method in mass spectrometry to measure the mass of ananalyte molecule, said method comprising the steps of: derivitizing asurface on a probe tip face with an affinity reagent having means forselectively bonding with an analyte molecule; exposing said derivitizedprobe tip face to a source of said analyte molecule so as to bond saidanalyte molecule thereto; placing the probe tip into one end of atime-of-flight mass spectrometer and applying a vacuum and an electricfield to form an accelerating potential within the spectrometer;striking the probe tip face within the spectrometer with a series oflaser pulses in order to desorb ions of said analyte molecules from saidtip; detecting the mass weights of the ions by their time of flightwithin said mass spectrometer; and displaying such detected massweights.
 3. The method according to claim 2 comprising additionallyapplying a desorption assisting matrix material to said probe tip facein association with said affinity reagent, said matrix material beingapplied in a manner so as not to interfere with said means on saidaffinity reagent for selectively bonding with said analyte molecules, aportion of said analyte molecules which are not desorbed from said probetip remaining chemically accessible for subsequent analytical procedureswithout the necessity for separating them from said matrix material. 4.The method according to claim 3 comprising additionally, removing saidprobe tip from said mass spectrometer; performing a chemical procedureon said portion of said analyte molecules so as to alter the chemicalcomposition of said portion of said analyte molecules; reinserting saidprobe tip with said chemically altered analyte molecules thereon; andperforming a subsequent mass spectrometry analysis to determine themolecular weight of said chemically altered analyte molecules.
 5. Themethod according to claim 2 wherein said affinity reagent is chemicallybonded to said face of said probe tip.
 6. The method according to claim2 wherein said affinity reagent is physically adhered to said face ofsaid probe tip.
 7. The method according to claim 2 wherein said affinityreagent is adapted to chemically bond to said analyte molecules.
 8. Themethod according to claim 2 wherein said affinity reagent is adapted tobiologically adhere to said analyte molecules.
 9. The method accordingto claim 2 wherein said analyte molecules are biomolecules and saidaffinity reagent is adapted to selectively isolate said biomoleculesfrom an undifferentiated biological sample.
 10. The method according toclaim 3 wherein said matrix materials are in the weakly acidic tostrongly basic pH range.
 11. The method according to claim 3 whereinsaid matrix materials have a pH above 6.0.
 12. The method according toclaim 2 wherein said face of said probe tip is formed of an electricallyinsulating material.
 13. A method of measuring the mass of analytemolecules by means of laser desorption/ionization, time-of-flight massspectrometry in which a matrix material is used in conjunction with saidanalyte molecules for facilitating desorption and ionization of theanalyte molecules, the improvement comprising: presenting the analytemolecules on or above the surface of the matrix material, whereby atleast a portion of the analyte molecules not desorbed in said massspectrometry analysis remain chemically accessible for subsequentanalytical procedures, in situ, on said probe tip, without the necessityfor separating said portion of said analyte molecules from said matrixmaterial.
 14. An apparatus for facilitating desorption and ionization ofanalyte molecules for analysis by mass spectrometry, said apparatuscomprising: a substrate; and an affinity reagent attached to saidsubstrate and having means for selectively bonding with said analytemolecules.
 15. The apparatus according to claim 14 wherein saidsubstrate comprises the surface of a probe tip for use in atime-of-flight mass spectrometry analyzer.
 16. The apparatus accordingto claim 14 wherein said affinity reagent is chemically bonded to saidsubstrate.
 17. The apparatus according to claim 14 wherein said affinityreagent is physically adhered to said substrate.
 18. The apparatusaccording to claim 14 wherein said affinity reagent is adapted tochemically bond to said analyte molecules.
 19. The apparatus accordingto claim 14 wherein said affinity reagent is adapted to biologicallyadhere to said analyte molecules.
 20. The apparatus according to claim14 wherein said analyte molecules are biomolecules and said affinityreagent is adapted to selectively isolate said biomolecules from anundifferentiated biological sample.
 21. The apparatus according to claim14 comprising additionally a matrix material deposited on said substratein association with said affinity reagent in a manner so as to notrender ineffective said means on said affinity reagents for selectivebonding with said analyte molecules.
 22. The apparatus according toclaim 21 wherein said matrix material is in the weakly acidic tostrongly basic pH range.
 23. The apparatus according to claim 21 whereinsaid matrix material has a pH above 6.0.
 24. The apparatus according toclaim 14 wherein said substrate is formed of an electrically insulatingmaterial.
 25. A method for preparing a surface for presenting analytemolecules for analysis by time-of-flight mass spectrometry, said methodcomprising: providing a substrate on said surface for supporting saidanalyte; derivitizing said substrate with an affinity reagent havingmeans for selectively bonding with said analyte; and depositing adesorption/ionization promoting matrix material on said substrate inassociation with said affinity reagent, said matrix material beingdeposited in a manner so as to not render ineffective said means on saidaffinity reagent for selectively bonding with said analyte.
 26. A methodfor preparing a surface for presenting analyte molecules for analysis,said method comprising: providing a substrate on said surface forsupporting said analyte; derivitizing said substrate with an affinityreagent having means for selectively bonding with said analyte; and ameans for detection of said analyte molecules bonded with said affinityreagent.
 27. The method according to claim 26 comprising additionallythe step of applying a detection material to said surface.
 28. Themethod according to claim 27 wherein such detection material comprises afluorescing species.
 29. The method according to claim 27 wherein suchdetection material comprises an enzymatic species.
 30. The methodaccording to claim 27 comprising additionally wherein such detectionmaterial comprises a radioactive species.
 31. The method according toclaim 27 comprising additionally wherein such detection materialcomprises a light-emitting species.